Signaling between neurons is accomplished, in large part, by chemical neurotransmission which involves the release of neurotransmitter from a presynaptic neuron, the detection of the transmitter on receptors located on both post- and presynaptic neurons, and the `uptake' of the transmitter by neurotransmitter sodium symporters (NSSs). NSSs are sodium-coupled membrane transport proteins that harness the pre- existing ion gradient to `pump' neurotransmitters into cells, up the concentration gradient. Because the removal of transmitter from the synaptic cleft and surrounding extracellular spaces is crucial to neurotransmission, dysfunction of NSSs underpins a number of neurological conditions and there are many important therapeutic agents, as well as illicit substances, that target NSSs, including antidepressants, anti- anxiety medications, amphetamines and cocaine, as examples. The goals of the research described in this grant application are to further our understanding of the biochemical mechanism of NSS function, to better understand how small molecule therapeutic agents and illicit substances interact with NSSs and to provide structure-based understanding of the consequences of selected naturally occurring mutations in NSS genes. The major focus of the research will be to study the human serotonin and dopamine transporters using the structural methods of single particle cryo electron microscopy (cryo-EM) and x-ray crystallography in combination with complementary biochemical, biophysical and computational methods. We are particularly interested in understanding, at the level of molecular detail, how NSSs isomerize from the outward facing conformations required to bind neurotransmitter to the inward facing conformations that are required to release transmitter. Moreover, we will elucidate the molecular contacts that underpin the interactions between key small molecules and NSSs to understand the principles of affinity and specificity, and we will also examine how key posttranslational modifications, such as phosphorylation, affect transporter conformation. The results from our studies will allow for a comprehensive understanding of the relationships between NSS structure and function and they will also provide an underpinning for further structure-based design of small molecules with potentially useful properties. Our studies will not only inform the immediate NSS field but, because NSSs are paradigm integral membrane transporter proteins, our studies will enlighten the much larger field of membrane transport.